Comparative Analyses of the Three-dimensional Structures and Enzymatic Properties of α, β, γ, and δ Isoforms of Ca2+-Calmodulin-dependent Protein Kinase II

Abstract

Ca2؉ -calmodulin-dependent protein kinase II (CaMkinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (␣, ␤, ␥, and ␦) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 Å revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their K m for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca 2؉ -calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (␥ > ␤ > ␦ > ␣) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (␤ > ␥ > ␦ > ␣). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform.1 is a major downstream effector of Ca 2ϩ signaling in eukaryotic cells. A rise in intracellular Ca 2ϩ concentration leads to binding of Ca 2ϩ ions to calmodulin (CaM), which binds to and activates CaM-kinase II. Upon activation, this enzyme has the ability to autophosphorylate, a process that confers Ca 2ϩ -independent activity upon the kinase (1) and greatly increases its affinity for CaM (2). Once activated, CaM-kinase II phosphorylates numerous target proteins and is involved in many cellular functions, including synaptic plasticity, synaptic vesicle mobilization, regulation of gene expression, regulation of smooth muscle contractility, and modulation of ion channel function (3-7). The fact that CaM-kinase II has so many potential substrates raises the question of the relationship between its activation and a specific response to a particular Ca 2ϩ signal. Possibly, the regulated expression of the multiple isoforms of CaM-kinase II confers these unique properties.CaM-kinase II is expressed from a family of four closely related genes, ␣, ␤, ␥, and ␦, each of which produces mRNA that can be alternatively spliced, giving rise to at least 30 different proteins (8, 9). The overall organization of each of the four kinase isoforms is similar: an N-terminal catalytic domain is followed by a regulatory domain that contains an autoinhibitory region and a CaM-binding site, and a C-terminal association domain, through which the subunits interact to assemble into holoenzymes (10). Between the CaM-binding domain and the association domain is a region termed the...

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